1. Field of the Invention
This invention is in the fields of molecular and cellular biology. More particularly, the invention is directed to mutants of the genes encoding Green Fluorescent Protein (GFP) and the proteins encoded by these mutants. The mutant GFPs are used to allow detection of eukaryotic and prokaryotic cells transfected or transformed with extrinsic genes, and to label proteins of interest to facilitate their localization within viable cells.
2. Related Art
Transfection of Foreign Genes
To study the function of a gene, a technique that is commonly employed is the transfer of the gene into a new cellular environment. This process, called xe2x80x9ctransfection,xe2x80x9d provides several advantages to the genetic scientist. For example, the cellular protein encoded by the gene can often be more easily studied by transferring the gene into a cell or organism that normally does not produce the protein, and then examining the effect of this protein on the host cell. The existence and function of regulatory genetic sequences (e.g., promoters, inhibitors and enhancers) may be elucidated by transfection of foreign genes into cells containing the regulatory sequences. The transfer of non-native or altered genes into a host cell also allows for large-scale production of the proteins encoded by the genes, a process upon which much of the current biotechnology industry is based. Transfection of plant embryos with foreign genes has provided genetically engineered plants that are more resistant to adverse environmental conditions or that are more nutritionally rich. Finally, gene transfer methods allow the introduction of new or mutated genes into whole organisms. This latter capability provides the opportunity for the construction of stable models of mammalian diseases, for large-scale production of proteins in the milk of transgenic lactating animals, and for the possibility of genetic therapy for certain diseases.
A variety of techniques has been used to transfect non-native genes into cells (reviewed in Sambrook, J., et al., Molecular Cloning, a Laboratory Manual, 2nd Ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, pp. 16.30-16.55 (1989); Watson, J. D., et al., Recombinant DNA, 2nd Ed, New York: W. H. Freeman and Co., pp. 213-234 (1992)). These techniques include biological methods such as the use of viruses (e.g., adenovirus or certain retroviruses for mammalian cells, baculovirus for insect cells and bacteriophages for bacterial cells) or bacteria (e.g., Agrobacterium for plant cells), chemical methods such as calcium phosphate precipitation, DEAE-dextran-mediated endocytosis or liposome-mediated transfection, and physical methods such as electroporation or direct microinjection. For transfection of mammalian cells, the techniques most commonly employed currently are virus-mediated transfection, lipofection and electroporation.
Detection of Gene Transfer
Regardless of the method used, however, simply attempting to transfect a cell does not guarantee that a majority (or even any) of the target cells will take up and/or express the exogenous DNA. Indeed, it has been suggested that the success rate of even the most optimal techniques used for transfection results in stable transfer of exogenous DNA is far less than 1% (Watson, J. D., et al., Recombinant DNA, 2nd Ed., New York: W. H. Freeman and Co., pp. 216, 218 (1992)). Thus, it is usually critical to determine which target cells have received and/or incorporated the gene(s) being transfected, for which a number of methodologies have been used.
Expression
The most obvious of these methods is to simply examine the target cells for expression of the exogenous gene. In this method, the transfected cells are grown in vitro and assayed for the presence of the protein encoded by the transferred gene. These assays are usually accomplished using immunological techniques such as Western blotting, ELISA or RIA. This type of technique is only useful, however, if the protein is produced in relatively high amounts (generally at the microgram level or above) and if suitable antibodies are available, neither of which is the case for some transfected genes.
In those cases where protein expression cannot be examined, incorporation of exogenous genes can be determined by assaying the target cells for production of the mRNAs corresponding to the transferred genes. One very common technique for this determination is Northern blotting (Alwine, J. C., et al., Proc. Natl. Acad Sci. USA 74:5350-5354, 1977), in which RNA molecules are isolated from cells, separated by gel electrophoresis and electroblotted onto a solid support (e.g., nitrocellulose or nylon). The solid support is then overlaid with radiolabelled cDNAs corresponding to the transfected gene, which hybridize on the solid support to their complementary mRNAs. After exposing the blot to photographic film, the samples containing the expressed transgene are easily determined. While this method is more sensitive than those directly measuring protein expression, Northern blotting still relies on actual expression of the gene by the target cells, which is not always the case.
Selection
Another method for determining gene transfer, alternative to directly measuring gene expression, is to examine the effect of the gene on the transfected cells. For example, some transfected genes will confer upon their host cells the ability to grow in selective culture media or under some other environmental stress which non-transfected cells cannot tolerate. Genes of interest are often engineered into sequences conferring, for example, antibiotic resistance upon the recipient cells. Transfectants with these constructs will thus carry not only the gene of interest but also the antibiotic resistance gene which allows them to grow in antibiotic-containing media. Since non-transfected cells will not possess this resistance, any cell able to grow in media containing antibiotic will contain the resistance marker (the so-called xe2x80x9cselectable markerxe2x80x9d) and the transgene that is linked to it. Selectable markers commonly used in such an approach are the neomycin (neo), ampicillin (amp) and hygromycin (hyg) resistance genes.
In the same way, selectable markers conferring on the transfected cells a metabolic advantage (e.g., ability to grow in nutrient-deficient media) have been used successfully. Examples of these types of selectable markers include thymidine kinase (Bacchetti, S., and Graham, F. L., Proc. Natl. Acad. Sci. USA 74:1590-1594 (1977); Wigler, M., et al., Cell 11:223-232 (1977)) and xanthine-guanine phosphoribosyltransferase (Mulligan, R. C., and Berg, P., Proc. Natl. Acad Sci. USA 78:2072-2076 (1981)), which impart to their recipients the ability to grow, using metabolic rescue pathways encoded by the marker genes, in media that inhibit vital metabolic pathways in non-transfected cells. Again, any cells able to grow in such media will contain the transgene linked to the marker gene.
Selection methods such as these often require weeks of culturing of the cells, continuously under selective pressure, to provide a relatively pure population of stable transfectants. Many uses of transfected cells, however, are conducted within hours of transfection, far too soon to determine transfection success using either the expression or selection methods described above. These types of applications are facilitated by a third approachxe2x80x94the use of xe2x80x9creporter genesxe2x80x9d.
Reporter Genes
Reporter genes are analogous to selectable markers in that they are co-transfected into recipient cells with the gene of interest, and provide a means by which transfection success may be determined. Unlike selectable markers, however, reporter genes typically do not confer any particular advantage to the recipient cell. Instead reporter genes, as their name implies, indicate to the observer (via some phenotypic activity) which cells have incorporated the reporter gene and thus the gene of interest to which it is linked. A number of reporter genes have been used, including those operating by biochemical or fluorescent mechanisms, each with its own advantages and limitations.
Biochemical Reporter Genes
Some commonly used reporter genes encode enzymes or other biochemical markers which, when active in the transfected cells, cause some visible change in the cells or their environment upon addition of the appropriate substrate. Two examples of this type of reporter sequence are the E. coli genes lacZ (encoding xcex2-galactosidase or xe2x80x9cxcex2-galxe2x80x9d) and gusA or iudA (encoding xcex2-glucuronidase or xe2x80x9cxcex2-gluxe2x80x9d); the former is often used as a reporter gene in animal cells (Hall, C. V., et al., J. Mol. Appl. Genet 2:101-109 (1983); Cui, C., et al., Trangenic Res. 3:182-194 (1994)), the latter in plant cells (Jefferson, R. A., Nature 342:837-838 (1989); Watson, J. D., et al., Recombinant DNA, 2nd Ed., New York: W. H. Freeman and Co., pp. 281-282 (1992); Hull, G. A., and Devic, M., Meth. Mol. Biol. 49:125-141 (1995)). These bacterial sequences are useful as reporter genes because the recipient cells, prior to transfection, express extremely low levels (if any) of the enzyme encoded by the reporter gene. When transfected cells expressing the reporter gene are incubated with an appropriate substrate (e.g., X-gal for xcex2-gal or X-gluc for xcex2-glu), a colored or fluorescent product is formed which can be detected and quantitated histochemically or fluorimetrically.
Another often-used reporter gene is the bacterial gene encoding chloramphenicol acetyltransferase (CAT), which catalyzes the addition of acetyl groups to the antibiotic chloramphenicol (Gorman, C. M., et al., Mol. Cell. Biol. 2:1044-1051 (1982); Neumann, J. R., et al., BioTechniques 5:444-446 (1987); Eastman, A, BioTechniques 5:730-732 (1987); Felgner, P. L., et al., Ann. N.Y. Acad Sci. 772:126-139 (1995)). After transfection, recipient cells are lysed and the lysates are incubated with radiolabelled chloramphenicol and an acetyl donor such as acetyl-CoA, or with unlabeled chloramphenicol and radiolabeled acetyl-CoA (Sleigh, M. J., Anal. Biochem. 156:251-256 (1986)). If expressed in the cells, CAT transfers acetyl groups to chloramphenicol, which is then easily assayed by chromatographic techniques, thereby giving an indication of the incorporation of the co-transfected gene of interest by the recipient cells.
Using reporter genes in this way, populations of cells, or even single cells, can be rapidly assayed for their incorporation of the exogenous gene linked to the reporter gene. Since they do not rely directly on the expression of the gene of interest, assays of transfection success using reporter genes are usually simpler and more sensitive than those measuring mRNA or protein production from the transgene (Watson, J. D., et al., Recombinant DNA, 2nd Ed., New York: W. H. Freeman and Co., p. 155 (1992)). However, the use of reporter genes is severely limited in that it usually requires sacrifice (fixation) of the cells prior to assay, and therefore cannot be used for assaying living cells or cultures. Thus, alternative means for determining the incorporation of the transgene in viable cells have been developed.
Fluorescent Reporter Genes
An example of viable reporter genes that are rapidly gaining widespread use are those that are fluorescence-based. These genes encode proteins which are either naturally fluorescent or which convert a substrate from nonfluorescent to fluorescent. Assays using this type of reporter gene are non-destructive and, owing to the availability of sophisticated fluorescence detection systems, are often more sensitive than biochemical reporter gene assays.
One example of a fluorescence reporter gene is the luciferin-luciferase system (Bronstein, I., et al., Anal. Biochem. 219:169-181 (1994)). This system utilizes the gene for luciferase, an ATPase enzyme isolated from fireflies (Gould, S. J., and Subramani, S., Anal. Biochem. 175:5-13 (1988)) and other beetles (Wood, K. V., et al., J. Biolumin. Chemilumin. 4:289-301 (1989)), or from certain bioluminescent bacteria (Stewart, G. S., and Williams, P., J. Gen. Microbiol. 138:1289-1300 (1992); Langridge, W., et al., J. Biolumin. Chemilumin. 9:185-200 (1994)). For use as a reporter gene, the luciferase gene is placed into a vector also containing the gene of interest, or separate vectors containing the luciferase gene and the gene of interest are mixed together. Cells are then transfected with the vector(s) and treated with the luciferase substrate luciferin which is rendered luminescent (and impermeant) intracellularly by the action of the luciferase. Cells containing the luciferase gene, and thus the gene of interest linked to it, can then be rapidly and sensitively observed using luminescence detectors such as luminometers.
To provide a further increase in sensitivity, attempts have been made to use genes from certain cyanobacteria which encode naturally fluorescent phycobiliproteins such as phycoerythrin and phycocyanin. These proteins are among the most highly fluorescent known (Oi, V. T., et al., J. Cell Biol. 93:981-986 (1982)), and systems have been developed that are able to detect the fluorescence emitted from as little as one phycobiliprotein molecule (Peck, K., et al., Proc. Natl. Acad Sci. USA 86:4087-4091 (1989)). Phycobiliproteins also have the advantage of being naturally fluorescent, thus eliminating the time-consuming steps of the addition of exogenous substrates for their detection as is required for luciferase and biochemical reporter genes. However, the phycobiliproteins have proven extremely difficult to engineer into gene constructs in such a way as to maintain their fluorescence (Heim, R., et al., Proc. Natl. Acad. Sci. USA 91:12501-12504 (1994)), and thus are not commonly used as reporter genes in assaying the transfection of mammalian cells.
Thus, the ideal reporter gene would encode a naturally fluorescent protein (for ease of use following transfection) that is highly fluorescent (for increased sensitivity) and easily engineered (for maintenance of fluorescence). Such a system has recently been developed, using the Green Fluorescent Proteins (GFPs) isolated from certain marine cnidarians.
GFP
Overview
GFPs are involved in bioluminescence in a variety of marine invertebrates, including jellyfish such as Aequorea spp. (Morise, H., et al., Biochemistry 13:2656-2662 (1974); Prendergast, F. G., and Mann, K. G., Biochemistry 17:3448-3453 (1978); Ward, W. W., Photochem. Photobiol. Rev. 4:1-57 (1979) and the sea pansy Renilla reniformis (Ward, W. W., and Cormier, M. J., Photochem. Photobiol. 27:389-396 (1978); Ward, W. W., et al., Photochem. Photobiol. 31:611-615 (1980)). The GFP isolated from Aequorea victoria has been cloned and the primary amino acid structure has been deduced (FIG. 1; Prasher, D. C., et al., Gene 111:229-233 (1992)) (SEQ ID NOs:1, 2). The chromophore of A. victoria GFP is a hexapeptide composed of amino acid residues 64-69 in which the amino acids at positions 64-67 (serine, tyrosine and glycine) form a heterocyclic ring (Prasher, D. C., et al., Gene 111:229-233 (1992); Cody, C. W., et al., Biochemistry 32:1212-1218 (1993)). Resolution of the crystal structure of GFP has shown that the chromophore is contained in a central xcex1-helical region surrounded by an 11-stranded xcex2-barrel (Ormo, M., et al., Science 273:1392-1395 (1996); Yang, F., et al., Nature Biotech. 14:1246-1251 (1996)). Upon purification, native GFP demonstrates an absorption maximum at 395 nanometers (nm) and an emission maximum at 509 nm (Morise, H., et al., Biochemistry 13:2656-2662 (1974);Ward, W. W., et al., Photochem. Photobiol. 31:611-615 (1980)) with exceptionally stable and virtually non-photobleaching fluorescence (Chalfie, M., et al., Science 263:802-805 (1994)).
While GFP has been used as a fluorescent label in protein localization and conformation studies (Heim, R., et al., Proc. Natl. Acad Sci. USA 91:1250-1254 (1994); Yokoe, H., and Meyer, T., Nature Biotech. 14:1252-1256 (1996)), it has gained increased attention in the field of molecular genetics since the demonstration of its utility as a reporter gene in transfected prokaryotic and eukaryotic cells (Chalfie, M., et al., Science 263:802-805 (1994); Heim, R., et al., Proc. Natl. Acad. Sci. USA 91:1250-1254 (1994); Wang, S., and Hazelrigg, T., Nature 369:400-403 (1994)). GFP has also been used in fluorescence resonance energy transfer studies of protein-protein interactions (Heim, R., and Tsien, R. Y., Curr. Biol. 6:178-182 (1996)). Since GFP is naturally fluorescent, exogenous substrates and cofactors are not necessary for induction of fluorescence, thus providing GFP an advantage over the biochemical, luminescent and other fluorescent reporter genes described above. Visualization of GFP fluorescence does not require the fixation steps necessary with biochemical reporters such as xcex2-gal and xcex2-glu, nor does it require extraction from the cell prior to assay as may be required with luciferase; thus, GFP is suitable for use in procedures requiring continued viability of transfected cells. In addition, since the GFP cDNA containing the complete coding region is less than 1 kilobase in size (Prasher, D. C., et al., Gene 111:229-233 (1992)), it is easily manipulated and inserted into a variety of vectors for use in creating stable transfectants (Chalfie, M., et al., Science 263:802-805 (1994)).
Despite these advantages, however, the use of wildtype GFP has a few limitations. For example, the excitation and emission maxima of wildtype GFP are not within the range of wavelengths of standard fluorescence optics (at which GFP demonstrates relatively low quantum yield (i.e., low intensity of fluorescence)). In addition, GFP shows low efficiency of transcription in mammalian cells upon transfection and is packaged into low-solubility inclusion bodies in bacteria (thus providing difficulty in purification). These limitations have been overcome to a limited extent via the introduction of selected point mutations into the sequence of wildtype GFP.
GFP Mutants
One of the earliest mutation studies of GFP, in which the tyrosine residue at position 66 in the wildtype protein (xe2x80x9cwt-GFPxe2x80x9d) was replaced with a histidine residue, resulted in a mutant protein which fluoresced blue instead of green when excited with ultraviolet (UV) light (Heim, R., et al., Proc. Natl. Acad. Sci. USA 91:1250-1254 (1994)). This mutant protein not only provided a capacity for two distinguishable wavelengths for use in studies comparing independent proteins and gene expression events, but also demonstrated that single point mutations in GFP could induce drastic changes in the photochemistry of the protein. Three other sets of specific point mutations have been shown to increase the excitation and emission maxima of GFP such that they fall well within the range of standard fluorescein optics (Ehrig, T., et al., FEBS Letts. 367:163-166 (1995); Delagrave, S., et al., Bio/Technology 13:151-154 (1995); Heim, R., and Tsien, R., Curr. Biol. 6:178-182 (1996)), thus permitting the use of GFP with standard laboratory fluorescence detection systems. The problem of low quantum yield by wt-GFP has been partially addressed by mutating the serine residue at position 65 to a threonine (xe2x80x9cS65Txe2x80x9d), either without (Heim, R., et al., Proc. Natl. Acad Sci. USA 91:12501-12504 (1994)) or with (Cormack, B., et al., Gene 173:33-38 (1996)) a concomitant mutation at position 64, or by mutating other residues in the non-chromophore region (Crameri, A., et al., Nature Biotech. 14:315-319 (1996)). The S65T mutation also appears to improve the rate of fluorophore formation in transfected cells by approximately four-fold over wt-GFP, thus allowing earlier and more sensitive detection of transfection with this mutant than with wt-GFP (Heim, R., et al., Proc. Natl. Acad. Sci. USA 91:1250-1254 (1994)). By combining the S65T mutation with a mutation at position 64 replacing phenylalanine with leucine, approximately 90% of the mutant GFP expressed in bacteria is soluble, thus improving protein purification and yields (Cormack, B., et al., Gene 173:33-38 (1996)). Another series of mutations results in a mutant fusion GFP consisting of linked blue- and green-fluorescing proteins which have proven useful in studies of protein localization, targeting and processing (Heim, R., and Tsien, R. Y., Curr. Biol. 6:178-182 (1996)). Analogously, chimeric constructs comprising GFP linked to other proteins have been used in studies of ion channel expression and function (Marshall, J., et al., Neuron 14:211-215 (1995)), and in organelle targeting studies where they have provided a means for selectively and distinctively labeling the organelles of living cells (Rizzuto et al., Curr. Biol. 6:183-188 (1996)). Finally, by combining the S65T mutation with other mutations throughout the nonchromophore regions of the wt-GFP gene, a xe2x80x9chumanizedxe2x80x9d mutant GFP (SEQ ID NOs:3, 4) has been produced that not only shows a significant increase in fluorescence intensity and rate of fluorophore formation over wt-GFP (via the S65T mutation) but also demonstrates a 22-fold increased expression efficiency in mammalian cells (Evans, K., et al., FOCUS 18(2):40-43 (1996); Zolotukhin, S., et al., J. Virol. 70:4646-4654 (1996)). This humanization was achieved via 92 base substitutions (in 88 codons) to the wt-GFP gene which were amino acid-conservative and which were made to provide a pattern of codon usage more closely resembling that of mammalian cells, as opposed to the jellyfish codon patterns found in the wt-GFP gene which are less efficiently translated in mammalian cells. A summary of these GFP chromophore mutants is presented in Table 1.
Despite some success in overcoming certain of the above-described limitations of GFPs, the sensitivity of GFP as a reporter gene (measured as percentage of positive cells) is not as high as that of standard biochemical reporter genes such as xcex2-gal (Evans, K., et al., FOCUS 18(2):40-43 (1996)). In addition, the use of GFP as a reporter gene or a protein tag requires the use of fluorescent excitation and emission optics, which increases user expense and which is more technically challenging than the use of visible or white light optics often used with standard reporters such as xcex2-gal. Thus, a need currently exists for additional GFP variants which are more highly fluorescent, humanized, rapidly expressed in mammalian cells, capable of being observed using standard white light optics, and which provide an increased level of sensitivity.
It is thus an object of the present invention to provide mutant GFP cDNAs and proteins. In one aspect, the invention relates to such mutant GFP cDNAs which, when transfected into prokaryotic. (e.g., bacterial) or eukaryotic (e.g., mammalian) cells, increase the sensitivity of detection (measured as percentage or number of positive cells). The present invention thus provides nucleic acid molecules encoding mutant GFPs, wherein the mutant GFPs have an amino acid sequence comprising an amino acid residue lacking an aromatic ring structure at position 64 and an amino acid residue having a side chain no longer than two carbon atoms in length at position 65. Preferably, (a) if the residue at position 64 is leucine then the residue at position 65 is not cysteine or threonine; (b) if the residue at position 64 is valine then the residue at position 65 is not alanine; (c) if the residue at position 64 is methionine then the residue at position 65 is not glycine; and (d) if the residue at position 64 is glycine then the residue at position 65 is not cysteine. The invention is particularly directed to such nucleic acid molecules encoding mutant GFPs wherein the amino acid residue at position 64 is alanine, valine, leucine, isoleucine, proline, methionine, glycine, serine, threonine, cysteine, alanine, asparagine, glutamine, aspartic acid or glutamic acid, most preferably cysteine or methionine. The invention is also particularly directed to such nucleic acid molecules encoding mutant GFPs wherein the amino acid residue at position 65 is alanine, glycine, threonine, cysteine, asparagine or aspartic acid, most preferably alanine. In particular, the invention provides nucleic acid molecules encoding mutant GFPs wherein the amino acid at position 64 is cysteine or methionine and the amino acid at position 65 is alanine, and nucleic acid molecules encoding mutant GFPs having an amino acid sequence as set forth in either SEQ ID NO:5 or SEQ ID NO:6.
In additional aspects, the invention provides mutant GFPs encoded by any of the above-described nucleic acid molecules, vectors (particularly expression vectors) comprising these nucleic acid molecules, host cells (prokaryotic or eukaryotic (including mammalian)) comprising these nucleic acid molecules or vectors, and compositions comprising plasmid pGreenLantern-2/A1 or plasmid pGreenLantern-2/A4. The invention also provides methods for producing a mutant GFP, comprising culturing the above-described host cells under conditions favoring the production of a mutant GFP and isolating the mutant GFP from the host cell. The invention also provides mutant GFPs produced by these methods, particularly wherein the mutant GFPs emit fluorescent light when illuminated with white light. The invention also relates to compositions comprising the above-described mutant GFPs.
The invention is further directed to kits for transfecting a host cell with the nucleic acid molecules encoding the present mutant GFPs, such kits comprising at least one container containing a nucleic acid molecule encoding a mutant GFP such as those described above, which preferably comprises plasmid pGreenLantern-2/A1 or plasmid pGreenLantern-2/A4. These kits of the invention may optionally further comprise at least one additional container containing a reagent, preferably comprising a liposome and most preferably LIPOFECTAMINE(trademark), for delivering a mutant GFP, nucleic acid molecule into a host cell.
The invention is further directed to kits for labeling a polypeptide with the present mutant GFPs, such kits comprising at least one container containing a mutant GFP such as those described above, preferably a mutant GFP having an amino acid sequence as set forth in SEQ ID NO:5 or SEQ ID NO:6. These kits of the invention may optionally further comprise at least one additional container containing a reagent for covalently linking this mutant GFP to the target polypeptide.
The fluorescence of all of the GFP mutants provided by the present invention is observable with fluorescein optics, making these mutant proteins amenable to use in techniques such as fluorescence microscopy and flow cytometry using standard FITC filter sets. In addition, the fluorescence of certain of the present GFP mutants, particularly those having amino acid sequences as set forth in SEQ ID NOs:5 and 6, is visible using standard white light optics (e.g., incandescent or fluorescent indoor lighting, or sunlight). The nucleic acid molecules and mutant GFPs provided by the present invention thus contribute improved tools for detection of transfection, for fluorescent labeling of proteins, for construction of fusion proteins allowing examination of intracellular protein expression, biochemistry and trafficking, and for other applications requiring the use of reporter genes.
Other preferred embodiments of the present invention will be apparent to one of ordinary skill in light of the following drawings and description of the invention, and of the claims.